Effective inactivation of pathogens in medical products has been a public health concern since it was discovered that previously unknown diseases can be spread quickly through the administration of therapies. Therefore, biotechnological products are coming under increasingly stringent standards intended to decrease the risk of transmitting agents by their use. Potential contaminants not only are a problem in the manufacture of blood products but also in the production of safe vaccines.
One of the most critical steps in the production of vaccines against pathogens, in particular viral vaccines, is viral inactivation. In the case of virus inactivation, formalin is the most frequently used inactivating agent in the manufacture of vaccines. The formalin inactivation step has been validated with established analytical procedures. However, the introduction of highly stringent quality control tests such as mammalian cell culture tests, e.g., the Vero safety test, has demonstrated evidence of residual infectivity in some cases. In an effort to eliminate this residual infectivity mechanical disruption of aggregates and/or filtration turned out to be unsuccessful.
As an alternative to formalin treatment, UV inactivation has been considered for integration into the manufacturing process. The use of ultraviolet irridation-inactivation for human vaccines has been demonstrated before. Milzer et al. (Am. J. Pub. Health (1954) 44:26-33) and Wolf et al. (JAMA (1956) 161:775-81) have reported on immunogenicity results from studies in humans where they used UV inactivated poliomyelitis vaccine. Poliovirus is an unenveloped picornavirus, with a positive single stranded RNA genome in a single segment. As the viral genome is more susceptible to UV-damage than viral surface antigens, in the case of polio te viral capsid proteins, UV-inactivation was shown to have little negative effect on the biochemical characteristics or immunogenicity of the product. The targets for UV inactivation are primarily nucleic acids in contrast to proteins which are targeted by formalin. By combining formalin and UV-inactivation, scientists tried to overcome the limitations of isolated UV-inactivation or formalin-inactivation, respectively, when inactivating the particularly resilient poliovirus. See, e.g., McLean, et al., “Experiences in the Production of Poliovirus Vaccines,” Prog. Med. Virol., vol 1, pp. 122-164 (1958.)
UV radiation technologies have a broad application range in food, pharmaceutical, cosmetics and beverage industry, and drinking water. UV disinfection is a physico-chemical process, wherein covalent bonds of the cyclic molecules of the purine and pyrimidine bases are disrupted by the excitation energy of the UV wavelength radiation, damaging the nucleic acids and the genetic information that they encode. Microorganisms such as bacteria and yeasts, etc., as well as viruses that are exposed to effective UVC (100 to 280 nm) radiation are inactivated within seconds. Consequently, successful disinfection depends on the reduction-equivalent irradiation dose. The mean microbicidal irradiation dose expressed in J/m2 is measured in the irradiation zone using a biodosimeter. However, UV-inactivation alone is not suitable for producing safe and effective vaccine.
Taylor et al. (J. Immunol. (1957) 79:265-75) describe the inactivation of poliomyelitis virus with a formalin and ultraviolet combination. Molner et al. (Am. J. Pub. Health (1958) 48:590-8) describe the formation of a measurable level of circulating antibodies in the blood of subjects vaccinated with ultraviolet-formalin inactivated polyomyelitis vaccine. Truffelli et al. (Appl. Microbiol. (1967) 15:516-27) report on the inactivation of Adenovirus and Simian Virus 40 Tumorigenicity in hamsters by a three stage inactivation process consisting of formalin, UV light and β-propiolactone. Miyamae (Microbiol. Immunol. (1986) 30:213-23) describes the preparation of immunogens of Sendai virus by a treatment with UV rays and formalin. None of the concepts described above has ever been adopted for general use in vaccine production, although there has been an ever-present need for the production of safe and efficacious vaccines on an industrial scale. In addition, none of the cited studies employed a safety test for determining the residual infectivity of the “inactivated viruses” that is as sensitive as the Vero safety test used in the present study.
Surprisingly, the inventors have found that a combination of formalin and UV inactivation steps can be utilized to fully inactivate virus in a bulk virus production in a high-volume throughput system for the manufacture of inactivated virus for vaccine preparation on an industrial scale. This has also been shown with a high-titer concentrated viral preparation as used in the examples, where no residual viral activity is detected using the very sensitive Vero cell culture test. The inventors have also surprisingly demonstrated that this method works particularly well for enveloped viruses, such as orthomyxoviruses, as compared to the unenveloped viruses, such as the polio picornavirus. This discovery has important implications for the rapid production of safe, highly immunogenic vaccines for emerging and rapidly changing viral diseases such as interpandemic and pandemic influenza strains.